Contact lens wear can predispose the healthy cornea to infection, most commonly by the gram-negative bacterium Pseudomonas aeruginosa. Research into the pathogenesis of corneal infection has mostly used a scratch injury model that bypasses the epithelial barrier, hindering our understanding of how bacterial interactions with it influence pathogenesis. Our lab recently developed a superficial injury model and imaging methods that for the first time enables subcellular localization of individual colonizing bacteria within whole mouse eyeballs. Previously, details of corneal epithelial cell/bacterial interactions could only be studied in cultured cells, which do not necessarily mimic in vivo infections. P. aeruginosa encodes a Type Three Secretion System (T3SS), a molecular syringe that injects toxins into host cell cytoplasm. Using cultured cells, this lab has shown that most P. aeruginosa corneal isolates of secrete the T3SS toxin ExoS, and named them invasive strains, because they can replicate inside cultured epithelial cells. In mouse corneal infections, they can also be seen inside cells of the corneal epithelium using transmission electron microscopy, and can be recovered even after treatment with membrane non-permeable antibiotics. Cultured cell studies show that ExoS can remodel the plasma membrane into a bleb niche wherein bacteria replicate. While blebs containing intracellular bacteria also occur during infection in vivo (seen using the new live imaging strategy), how frequently, when, and where invasion occurs, whether intracellular bacteria replicate within native corneal epithelium, and if/how these phenomena contribute to pathogenesis is to be established. The hypothesis is that for an invasive strain, a significant percentage of bacteria colonizing the corneal epithelium in situ become intracellular in all three cellular layers, that intracellular localization activates the bacterial T3SS, and tha activation of the T3SS enables intracellular replication. Unpublished data supporting this show that the T3SS contributes to epithelial traversal within the mouse cornea, while data I collected using cultured cells show the T3SS is triggered only after bacteria are internalized, the later being a paradigm shift. Using the in situ mouse eye model, Aim 1 will determine bacterial distribution during corneal epithelial traversal, including intra/extracellular spaces and subcellular localization, Aim 2 will examine the role of T3SS activation and delivery of exotoxins into epithelial cell cytoplasm, while Aim 3 will determine sites of intracellular replication and ue mutant P. aeruginosa lacking T3SS components to determine their contribution. This training opportunity will allow me to build on a foundation in bacterial toxin biochemistry by providing experience with live bacteria, animal models, state of the art imaging technologies, and virulence factor regulation in vivo. The project will explore the significance of P. aeruginosa internalization, while determining how the T3SS contributes to traversal of the corneal epithelium. Given that epithelial traversal is an early step in infection, this research could leadto strategies for preventing infection before pathological processes are initiated.